The malarial parasite infects 10% of the world population and is responsible for 2-4 million deaths in over 80 nations each year. The wide-spread emergence of drug resistance to the parasite severely limits treatment options, and there is a clear need to develop new anti-malarial agents. Unlike mammalian cells, the malaria parasite cannot salvage preformed pyrimidine bases or nucleosides, and pyrimidines are exclusively synthesized by the de novo pathway. The importance of the pyrimidine biosynthetic pathway to the survival of the malarial parasite is demonstrated by the finding that dihydrofolate reductase is a validated target for the treatment of the parasite. For these reasons any of the key enzymes in the pathway are potential drug targets. However, several factors make the fourth step, catalyzed by dihydroorotate dehydrogenase (DHODH) particularly attractive. DHODH has been successfully exploited as a drug target for the treatment of rheumatoid arthritis. The enzyme has been well characterized from a number of bacterial and eukaryotic sources. X-ray structures are available for several family members, and a larger number of inhibitors have been generated. The inhibitor and crystallographic data suggest that the enzymes from different species have very divergent inhibitor binding sites, and species selective inhibitors has been reported for human and several bacterial enzymes. The aims of this grant are to validate DHODH as a target for treatment of the malarial parasite, and to identify potent and selective inhibitors of the enzyme that have activity against the parasite. The proposed studies will be broad ranging and will require the expertise and resources of two laboratories to accomplish. The studies include: 1) biochemical and structural characterization of recombinant malarial DHODH, 2) synthesis of a small molecule combinatorial library that will be screened for malarial DHODH inhibitors, 3) testing of identified inhibitors for antimalarial activity to develop structure/activity relationships, 4) comparison of the metabolic consequence of gene knockout to the effects of inhibitors by microarray analysis, and 5) generation of parasites that are resistant to DHODH inhibitors and analysis of mutations found in DHODH to determine the functional consequence on enzyme activity, and to correlate these changes to drug resistance by transformation into sensitive parasites.